Clinical and laboratory evaluation
The severity of all participants’ clinical course was determined on each hospital day using a modified Wang bronchiolitis severity score, configured by the replacement of patients’ general condition with oxygen saturation13. Oxygen saturation was considered as a more reliable measurement than the patients’ general condition, which was recorded by different physicians at the time of admission. Moreover, data support the necessity of oxygen saturation measurement for evaluating the severity of bronchiolitis14, while general condition may be the component of Wang score which presents the highest correlation with oxygen saturation15. Modified Wang score ranges from 0-12 with 12 being the most severe. Scoring was obtained as follows: respiratory rate (0:<30/min, 1:30-45/min, 2:46-60/min, 3:>60/min), use of accessory muscles (0:none, 1:intercostal recession, 2:trachea-sternal recession, 3:severe recessions with nasal flaring), room air oxygen saturation (0:> 95%, 1:90-94%, 2:<90%, 3:<85%), and the presence and extent of wheezing (0:none, 1:terminal expiration with stethoscope, 2:entire expiration and inspiration with stethoscope, 3:inspiration and expiration without stethoscope). The length of hospitalization, O2supplementation, intravenous (IV) fluid requirement, nasogastric (NG) feeding, use of any medication and Intensive Care Unit (ICU) admission were noted.
Serum 25(OH)D levels, as well as cathelicidin and β-defensin-2 levels in the respiratory secretions, were measured in the Research Laboratory of the 2nd Department of Pediatrics, National and Kapodistrian University of Athens.
Βlood samples were centrifuged within one hour of blood sampling. Serum samples were frozen at -20°C until further analysis. Quantitative measurement of serum 25(OH)D levels was carried out using the following ELISA laboratory kit: 25(OH) -Vitamin D direct ELISA Kit (Immunodiagnostik AG). Competitive ELISA technique with selected monoclonal antibody recognizing 25(OH)D was used.
Μeasurements of AMPs and total protein were performed at the same time for each nasal secretion sample in order to normalize the AMPs values. Nasal secretions were collected by vacuum-aided suction, without chemical stimulation, in order to avoid introducing foreign substances into the nasal fluids. Gentle manipulation of a narrow rubber-tipped vacuum device inside the nasal passageways mildly stimulated nasal secretions. Nasal samples were handled with Aprotinin (0.6TIU per ml of sample) immediately after collection in order to thwart the action of proteolytic enzymes. Secretions were stored at −20°C.
Quantitative analysis of AMPs in nasal secretions were carried out using the following ELISA Test Kits: Human Beta Defensin-2 (EK-072-37, Phoenix Pharmaceutical) and Human LL-37 (HK321-01, Hycult Biotechnology) according to manufacturer’s instructions. The specificity of the kit to both human β-defensin-2 and LL-37 is 100%. All ELISA measurements were carried out in duplicate and included internal standards used to construct standard curves for analyte concentration assessment.